Lab 4: Cell Division – Mitosis and Meiosis
How do eukaryotic cells divide to produce genetically identical cells or to produce gametes
with half the normal DNA?
Background:
One of the characteristics of living things is the ability to replicate and pass on genetic
information to the next generation. Cell division in individual bacteria and archaea usually occurs by
binary fission. Mitochondria and chloroplasts also replicate by binary fission, which is evidence of the
evolutionary relationship between these organelles and prokaryotes.
Cell division in eukaryotes is more complex. It requires the cell to manage a complicated process
of duplicating the nucleus, other organelles, and multiple chromosomes. This process, called the cell
cycle, is divided into three parts: interphase, mitosis, and cytokinesis. In the first growth phase (G1), the
cell grows and prepares to duplicate its DNA. In the synthesis phase (S), the chromosomes are
replicated. In the second growth phase (G2), the cell prepares to divide. In mitosis, the duplicated
chromosomes are separated into two nuclei. In most cases, mitosis is followed by cytokinesis, when the
cytoplasm divides and organelles separate into daughter cells. This type of cell division is asexual and
is important for growth, renewal, and repair of multicellular organisms.
Cell division is tightly controlled by complexes made
of several specific proteins. These complexes contain enzymes
called cyclin-dependent kinases (CDKs), which turn on or off
the various processes that take place in cell division. CDK
partners with a family of proteins called cyclins. One such
complex is mitosis-promoting factor (MPF), sometimes called
maturation-promoting factor, which contains cyclin A or B
and cyclin-dependent kinase (CDK). CDK is activated when it
is bound to cyclin, interacting with various other proteins
that, in this case, allow the cell to proceed from G2 into
mitosis. The levels of cyclin change during the cell cycle. In
most cases, cytokinesis follows mitosis.
As shown in Figure 2, different CKs are produced during the
phases. The cyclins determine which processes in cell division
are turned on or off and in what order by CDK. As each
cyclin is turned on or off, CDK causes the cell to progress
through the stages in the cell cycle.
Cyclins and CDKs do not allow the cell to progress through its cycle automatically. There are
three checkpoints a cell must pass through the G1 checkpoint, G2 checkpoint, and the M-spindle
checkpoint. At each of the checkpoints, the cell checks that it has completed all of the tasks needed and
is ready to proceed to the next step in its cycle. Cells pass the G1 checkpoint when they are stimulated
by appropriate external growth factors for example, platelet-derived growth factor (PDGF) stimulates
cells near a wound to divide so that they can repair the injury. The G2 checkpoint checks for damage
after DNA is replicated. And if there is damage, it prevents the cell from going into mitosis. The Mspindle (metaphase) checkpoint assures that the mitotic spindles or microtubules are properly attached
to the kinetochores (anchor sites on the chromosomes). If the spindles are not anchored properly, the
cell does not continue on through mitosis. The cell cycle is regulated very precisely. Mutations in cell
cycle genes that interfere with proper cell cycle control are found very often in cancer cells.
Figure 3 illustrates how the chromosomes move during mitosis. It is important for you to model
how the duplicated chromosomes align, separate, and move into new cells.
The stages of mitosis:
 Prophase. During prophase, the chromosomes supercoil and the fibers of the spindle apparatus
begin to form between centrosomes located at the pole of the cells. The nuclear membrane also
disintegrates at this time, freeing the chromosomes into the surrounding cytoplasm.
 Prometaphase. During prometaphase, some of the fibers attach to the centromere of each pair
of sister chromatids and they begin to move toward the center of the cell.
 Metaphase. At metaphase the chromosomes have come to rest along the center plane of the cell.
 Anaphase. During anaphase, the centromeres split and the sister chromatids begin to migrate
toward the opposite poles of the cell.
 Telophase. During telophase, the chromosomes at either end of the cell cluster begin to cluster
together, which facilitates the formation of a new nuclear membrane. This also is when
cytokinesis occurs, leading to two separate cells. One way to identify that telophase has begun
is by looking for the formation of the cell plate, the new cell wall forming between the two cells.
Learning Objectives: The objectives of this lab exercise are for you to
• Better understand the process and stages of mitosis.
• Prepare your own specimens of onion root in which you can visualize all of the stages of mitosis.
• Apply an analytical technique by which the relative length of each stage of mitosis can be
estimated.
I - Viewing mitosis in onion root tips.
Why use onion roots for viewing mitosis?
• The roots are easy to grow in large numbers.
• The cells at the tip of the roots are actively dividing, and thus many
cells will be in stages of mitosis.
• The tips can be prepared in a way that allows them to be flattened on
microscopes slide (“squashed”) so that the chromosomes of
individual cells can be observed.
• The chromosomes can be stained to make them more easily observable.
Regions of Onion Root tips
There are three cellular regions near the tip of an onion root.
1. The root cap contains cells that cover and protect the
underlying growth region as the root pushed through the soil.
2. The region of cell division (or meristem) is where cells are
actively dividing but not increasing significantly in size.
3. In the region of cell elongation, cell are increasing in size, but
not dividing.
Region of cell
elongation
Region of cell
division
Protective
root cap
Viewing Chromosomes
Chromosomes generally are not visible as distinct entities in nondividing
cells, since the DNA is uncoiled, but the process of mitosis is facilitated by supercoiling of the
chromosomes into a highly compacted form. Supercoiled chromosomes can be visualized in cells,
particularly if they are treated with a DNA-specific stain, such as the Feulgen stain.
Procedure for preparing root tip squashes
You will be provided with roots that have been previously harvested and treated with a fixative to
stabilize the cells.
The first step will be to ‘soften’ the roots so that they later can be spread on a
microscope slide.
1. Using scissors, cut 2 roots tips about 1 cm long, and transfer them into a
plastic micro-tube. (One of the roots will be an extra one.)
2. Fill the tube about 2/3 full with 1N HCl from a dropper bottle.
*** Caution: Work with the HCl carefully, it is a strong acid. ***
3. Place the tube in a 60OC water bath, and allow the roots to incubate for 12 minutes.
4. After the 12 minute incubation period, remove the tube from the water bath.
Rinse the roots in H2O.
1. Using forceps, carefully transfer the root tips to a small petri plate.
2. Using a plastic ‘squeeze’ pipet, carefully remove the HCl from the micro-tube and transfer it
to the “discard flask”.
3. Rinse the root tips 3 times with water from the dropper bottle, disposing of the rinses in
the discard flask.
Staining the chromosomes.
1. After removing the water from the third rinse, cover the root with the Feulgen stain.
*** Caution: Although the Feulgen stain does not appear colored, it
will strongly stain skin and clothing. ***
2. Incubate the roots in the stain for 12 minutes. During this time the very
tip of the root will begin to turn red as the DNA stains the numerous
small actively dividing cells at the tip.
Remove the stain and again rinse the roots.
1. Using a plastic ‘squeeze’ pipet, carefully remove the Feulgen stain
and discard it in the discard flask.
2. Again, rinse the root tips 3 times with water.
Preparing the root tip squash.
1. Transfer a root to the center of a clean microscope slide and add a drop
of water.
2. Using a razor blade cut off most of the unstained part of the root,
and discard it. Return the used razor blade to the material station in
the box marked “RAZORS”
3. Cover the root tip with a cover slip, and then carefully push down on the
cover slide with the wooden end of a dissecting probe. Push hard, but do
not twist or push the cover slide sideways. The root tip should spread out
to a diameter about 0.5 – 1 cm.
Observations of onion root tip squash.
Scan the microscope under the 10x objective.
Look for the region that has large nuclei
relative to the size of the cell; among these cells
will be found cells displaying stages of mitosis.
Examples are shown in the figure to the right.
Switch to the 40X objective to make closer
observations. Since prophase and
prometaphase are difficult to distinguish,
classify all these cells as prophase. Identify 100
cells and record your observations in the table
provided.
Interphase
Metaphase
Early Prophase
Anaphase
MidProphase
Telophase
II - Estimating the relative length of each stage of mitosis.
For this procedure, we will use permanently mounted slides of onion roots. These slides are
prepared by slicing the roots into thin sections, mounting them on microscope slides, staining, and
then mounting under cover slip.
Mitosis and the cell cycle
While making your observations, consider the
relative number of cells actually involved in mitosis. Some
of these cells are still involved in the cell cycle, which
encompasses all of the processes involved in cell
replication. Cell that are actively dividing but not yet in
mitosis are said to be in interphase, during which time the
DNA is copied and the cell is otherwise preparing for
replication. Some root cells have ceased dividing and are
only increasing in size, whereas others have reached their
final, mature size and function, and are said to be in the
GO stage.
Procedure for determining the length of the stages of mitosis
Locate the meristem region of the root tip.
1. Starting under the 10x objective, find the region of active cell division.
2. Switch to the 40x objective and begin observations at the lower end of this region.
Recording data
Students should take turns as observer and recorder. The observer should
call out the stage of mitosis of each cell to be tallied by the recorder in the
results table. Roles should be switched for the second slide. Since prophase
and prometaphase are difficult to distinguish, classify these cells as
prophase. Only count as prophase cells that contain distinctly visible
chromosomes.
1. Systematically scan the root tip moving upward and downward
through a column of cells.
2. Tally each cell in a stage of mitosis that you observe, being careful not
to record the same cell twice. Tally the stages of 100 cells.
3. Tally numbers in the table below. Each group member should tally
cells from a different slide.
Calculations
1. Pool your data with that of the class, and then record the class totals in the table provided
below.
2. Calculate the percentage of cells in each stage.
3. The relative time span of each stage is equivalent to the percentage of cells found in that stage.
Pre-Lab 4: Mitosis and Cell Division
Name ___________________
1. What Safety Precautions must be taken during the lab?
______________________________________________________________________________
______________________________________________________________________________
2. What materials will be used during Experiment I?
______________________________________________________________________________
______________________________________________________________________________
3. What regulates the cell cycle, and why is precise regulation important in cell division?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
4. What are the 3 phases of the Cell Cycle?
______________________________________________________________________________
5. Identify all 3 Cell Cycle Checkpoints and describe why each is necessary.
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
6. Briefly describe what you will look for to identify the different stages of Mitosis
a. Interphase ___________________________________________________________
_____________________________________________________________________
b. Prophase _____________________________________________________________
_____________________________________________________________________
c. Metaphase ___________________________________________________________
_____________________________________________________________________
d. Anaphase ____________________________________________________________
_____________________________________________________________________
e. Telophase ___________________________________________________________
____________________________________________________________________
Relative length of stages of mitosis: Tally the results of your cell counts and calculate percentages.
Your tally
Percent of Total
Interphase
Prophase
Metaphase
Anaphase
Telophase
Totals
100
Tally the results of your cell counts from the PREPARED SLIDES and then calculate percentages.
Your tally
Class Totals
Percent of Total (Class)
Interphase
Prophase
Metaphase
Anaphase
Telophase
Totals
100
Post-Lab Questions:
1. Based upon the class results, order the stages of mitosis from shortest (1) to longest (4).
After the longest and shortest stage, give a brief explanation of why that stage may have
that time period.
Prophase ___
Metaphase ___
Anaphase ___
Telophase ___
2. Many of the cells of the root meristem are not undergoing mitosis, rather they are in a stage
called _________________________. Based upon the interpretations made above, interphase
appears to be much ____________________________ (shorter / longer) than mitosis. What
processes occur in interphase cell prior to the onset of mitosis?
3. Once Mitosis ends the cell will go through _____________________________ and enter the
___________________________ stage. Why is it incorrect to say that these cells are “resting”?
4. Calculate a chi-squared analysis of each phase (we will go over this in class)